NK cells recognize virally infected and malignant cells and respond by direct target cell killing and the production of inflammatory cytokines. In humans, we have shown that adoptive transfer of NK cells can induce remission in patients with refractory acute myelogenous leukemia (AML) and that hematopoietic cell transplantation (HCT) can cure AML. Although clinical success is seen in both settings, NK cell survival and function are often suboptimal after transplant, resulting in cancer relapse. We have shown that AML patients who reactivate latent human cytomegalovirus (HCMV) post-HCT reconstitute more terminally differentiated NK cells with potent effector function relative to those who do not reactivate HCMV or are HCMV seronegative. Several clinical studies have also recently shown that HCMV reactivation early after HCT is associated with reduced relapse risk. The molecular basis for enhanced NK cell differentiation and function in response to HCMV is unknown. We have extensive preliminary data showing that, in healthy HCMV seropositive donors and in transplant recipients that experience HCMV reactivation, populations of NK cells lacking the proximal signaling molecules FcR1g, SYK and EAT-2 expand. These novel NK cells have a genome wide epigenetic profile markedly similar to that of effector CD8+ T cells with a striking microRNA signature. They are present at stable frequencies over the course of at least one year, and they exhibit enhanced function in response to signaling through CD16. These cells appear to represent a human equivalent to adaptive or memory NK cells in mice that respond specifically to mouse cytomegalovirus (MCMV). Because of their functional and survival properties, there is considerable interest in being able to utilize them for immunotherapy. In this proposal, we aim to study the role of microRNAs in the differentiation of adaptive human NK cells upon HCMV reactivation in NK cells from our unique cohort of transplant patients using an unbiased, cutting-edge deep-sequencing approach. We will test the hypothesis that signaling through activating receptors is required for the expansion of adaptive NK cells and determine the role of pentameric HCMV protein complexes and high-affinity antibodies to these complexes in triggering expansion. We have preliminary data suggesting that the Notch/TCF-1 and Wnt signaling pathways are selectively induced in HCMV-induced adaptive NK cells, and we will study the role of both pathways in the development and differentiation of adaptive NK cells in depth using our well defined transplant cohorts and in vitro assays. We will also combine our recently developed xenogeneic model for adoptive human NK cell transfer with a new model for systemic HCMV infection in mice to study HCMV-induced adaptive NK cell survival, homing and function in vivo. Our team of leading basic and clinical NK cell biologists, HCMV experts and mouse immunologists are well positioned to perform these studies and translate them to therapies that can prime NK cells for potent function and prolonged survival for clinical use in the treatment of cancer.